Protein%203-D%20structure:%203o%20and%204o%20structure%20and%20protein%20folding.

1

• Protein 3-D structure 3o and 4o structure and
  protein folding.

2
3o Structure

• third level of protein organization
• folding of polypeptide chain causes 2o structures
  to interact
• formation of motifs and domains

3
Proteins with similar 1o structure also have
similar 3o structure
tuna 1 GDVAKGKKTFVQKCAQCHTVENGGKHKVGPNLWGLFGRK
TGQAEGYSYTDANKSKGIVWNyeast 1
GSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTD
ANIKKNVWDErice 1 GNPKAGEKIFKTKCAQCHTVDKGAGHKQ
GPNLNGLFGRQSGTTPGYSYSTANKMAVIWEEtuna 61
ETLMEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATSyeast
61 NNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACEric
e 61 NTLYDYLLNPKKYIPGTKMVFPGLKKPQERADLISYLKEATS

4
Common Motifs

• Motif Composition
• Helix-loop-helix all alpha-helix
• Coiled-coil
• Helix bundle
• Beta meander all beta sheet
• Greek key
• Beta-alpha-beta mixed alpha/beta

5
Motifs Combine to form Domains

• Domains are independent folding units in a 3o
  structure of a protein
• Individual domains have specific function

Parallel twisted sheet

• Hydrophobic interactions are the major driving
  force in folding domains

Alpha/beta barrel
6
Protein family members share common domain
structures
lactate dehydrogenase
malate dehydrogenase
NAD
NADH
7
4o Structure

• Quaternary structure describes the organization
  of subunits in a protein with multiple subunits
  (oligomeric protein)
• Can have homo-multimers or hetero-multimers

a2b2
a2bg
8
4o Structure

• Determine molecular weight of native protein by
  gel permeation chromatography
• Determine molecular weight of individual subunits
  by SDS-PAGE
• Can use the information to determine subunit
  composition

If. Native protein 160,000 daltons and
a-Subunit 50,000 daltons b-Subunit 30,000
daltons Then Protein can have a2b2 structure
9
4o Structure

• Subunits held together by non-covalent
  interactions
• Oligomeric protein is more stable than
  disassociated subunits
• Active site often made up of AA residues from
  different subunits
• 4o and 3o structure is often affected by ligand
  (substrate or inhibitor) binding. Important in
  enzyme regulation

10
Protein denaturation

• Denaturation disruption of native conformation
• Heat commonly used to denature proteins
• Tm temperature where 50 folded/50 unfolded.
• Typical Tm 40-60oC
• Tm for thermophiles gt100oC (Taq DNA
  polymerase)
• Chemical denaturants Chaotrophic agents Urea,
  KCN detergents SDS

11
Protein Folding

• Ribonuclease A (RNase A) will refold to native
  structure spontaneously (1 minute)
• gt1050 possible conformations
• If 10-13 sec per conformation would take 1030
  years to sample enough to determine structure
• How do proteins fold so quickly?

12
Factors driving protein folding

• Conformational entropy AB ? C decreases entropy
  (unfavorable)
• Non-covalent interactions give favorable enthalpy
  value
• Hydrophobic effect increases entropy by freeing
  water (favorable)

DG DH - TDS
-

13
Protein Folding

• Structures of globular proteins are not static
• Proteins breathing between different
  conformations
• Proteins fold towards lowest energy conformation
• Multiple paths to lowest energy form
• All folding paths funnel towards lowest energy
  form
• Local low energy minimum can slow progress
  towards lowest energy form

14
Pathway of Protein Folding
1) Nucleation of folding - Rapid and reversible
formation of local 2o structures form
2) Formation of domains (Molten Globular
intermediates) through aggregation of local 2o
structures
3) Domain conformations adjust to form native
protein
15
Chaperonins

• Protein complexes that promote protein folding
• Chaperonins dont determine native structure
• Prevent misfolding and aggregation of protein
• Sequesters unfolded protein from other proteins
• Require ATP for protein binding, after ATP
  hydrolysis native protein released
• Thought to bind unfolded regions of protein

16
Disulfides Bonds

• Stabilize native structure
• Formed after native conformation achieved
• Abundant in secreted proteins but not in
  intracellular proteins
• Protein disulfide isomerase catalyzes reduction
  of incorrect disulfide linkages